Project Summary/Abstract Although treatment for children with high-risk metastatic neuroblastoma has improved significantly in the past 20 years, only 45% of these patients become long-term, disease-free survivors, and bone marrow is a frequent site of resistant or recurrent disease. Improvement in survival necessitates development of effective new therapies that target biologically risk-stratified subgroups of patients and also development of effective biomarker assays that assess response and provide surrogates for long-term treatment benefit. Specific Aims: 1) Determine if quantifying "tumor load" in bone marrow and blood with a TaqMan(R) Low Density Array (TLDA) assay for 5 neuroblastoma associated genes improves response evaluation and prediction of outcome compared to immunocytology and clinical evaluations. 2) Determine if bone marrow and blood "microenvironment" gene expression signatures predict outcome and if combining these with tumor load data further improves prediction of outcome. 3) Determine if combining data from the TLDA tumor load/microenvironment assay for blood and marrow and a TLDA predictive gene expression assay for primary tumor from the same patient improves prediction of outcome compared to either assay alone. Research Design and Methods: We developed a new TLDA assay that can simultaneously quantify expression of five neuroblastoma genes ("tumor load") and 38 "microenvironment" genes representing normal blood and marrow cells. This assay quantifies expression of CHGA, DCX, DDC, PHOX2B, and TH so that one tumor cell among 106 normal bone marrow or blood mononuclear cells can be identified. Preliminary studies indicate that "tumor load" in PBSC and bone marrow that does not have detectible tumor cells by immunocytology correlates with outcome. In a separately funded project, we have developed another TLDA assay that quantifies a prognostic 14-gene expression signature in primary MYCN non-amplified tumors to predict outcome, and another signature is being developed for MYCN amplified tumors. We shall extend and validate our preliminary studies by testing 3,000 viably cryopreserved specimens (all have been tested by immunocytology) from three independent cohorts of patients who have been/are being enrolled in six different studies of the Children's Oncology Group. Analysis will take into account when the specimen was obtained (diagnosis and during and at the conclusion of therapy) and treatment (induction, consolidation, and post-consolidation therapies) in evaluating response and predicting outcome compared to immunocytology and standard clinical assessments. Tumor load and microenvironment data for blood and marrow will be combined with gene expression signature data for primary tumors to determine their relationship and their ability to improve prediction of outcome for clinical decision making. Summary: It is anticipated that our new TLDA assay for neuroblastoma cells in marrow and blood will improve prediction of outcome and hence to development of more effective therapy for patients with high-risk metastatic neuroblastoma. PUBLIC HEALTH RELEVANCE: Project Narrative Although treatment has steadily improved in the past 20 years for children with high-risk metastatic neuroblastoma (stage 4), only 45% of these patients survive long-term, and bone marrow is a frequent site of resistant or recurrent disease. We have developed a robust biomarker assay that simultaneously quantifies "tumor load" by expression of tumor cell genes and "microenvironment" by expression of normal marrow and blood cell genes. We anticipate that this assay will become an integral biomarker tool for evaluating response to new therapeutic strategies and for predicting outcome of children with high-risk neuroblastoma.